A small population of CD133 (a stem cell marker) positive "tumor stem cells" has been recently shown to embody the tumorigenic phenotype of human glioblastomas. In preliminary studies, we have shown 1) that 100% of CD133-positive stem cells in fresh surgical isolates of human glioma express the gliogenic transcription factor OLIG2, 2) that OLIG2 is essential for tumor formation in a mouse model of glioma that emulates human gliomas at a genetic level, 3) that the cell cycle inhibitor P21 is derepressed in OLIG2 deficient mouse neural stem cells by expression analysis, and 4) that OLIG2 binds to P21 promoter in human glioblastoma by chromatin immunoprecipitation thus linking OLIG2 to the cell cycle regulatory apparatus. My objectives are to test the hypothesis that OLIG2 is essential for the malignant growth of human CD133-positive human glioma stem cells in in vitro and in vivo model systems and to elucidate the mechanism by which OLIG2 affects tumor stem cells. The Specific Aims are as follows: Aim 1 is to test the prediction that P21 expression segregates from OLIG2 in diffuse human gliomas by in situ hybridization and immunofluorescent methods on surgical isolates. Aim 2 is to test the prediction that suppression of OLIG2 will stimulate the expression of P21 in human glioma stem cells and suppress their malignant phenotype using RNAi lentiviral vectors against OLIG2. Aim 3 is to test the prediction that P21 is epistatic to OLIG2 for maintenance of the malignant phenotype by determining whether ectopic P21 can suppress the transformed phenotype of CD133-positive human glioma stem cells as per Aim 2. Aim 4 is to expand our repertoire of direct genetic targets of OLIG2 through chromatin immunoprecipitation-tiled array ("ChlP-on-chip") analysis of OLIG2 interactions in human CD133-positive stem cells. Over the term of this K08 I will learn and develop new techniques, approaches, and methodologies that will allow me to develop into an independent investigator. My long-range goals are to study how normal neuro-developmental mechanisms can be subverted to form brain cancer and to develop rational approaches to target these mechanisms to improve survival of our brain tumor patients. These studies may additionally provide insights into other aspects of clinical neurobiology given the broad involvement of glia in many neurological disorders. Relevance: Glioblastoma is the most common type of brain tumor affecting young adults in the prime of their social and family life causing tremendous morbidity and mortality for which there is no cure. The goal of this application is to elucidate the role of OLIG2 in human glioma stem cells with the aim of discovering a new target(s) for the treatment of this devastating cancer.